Team:RHIT/Description

Project Description

The yeast Saccharomyces cerevisiae is used industrially to produce valuable products via fermentation. Many of these products are made under anaerobic conditions, when the electron transport chain in mitochondria lacks the terminal electron acceptor and oxidative phosphorylation ceases (Nevoigt 380). We propose that the production of fermentation products and secondary metabolites could be optimized by purposefully regulating aerobic respiration during industrial fermentations. We intend to address our objective by manipulating the expression of mitochondrial ribosomal protein S12 (MRPS12). This protein, which is encoded by the nuclear MRPS12 gene (systematic name, YNR036C) is essential for the function of mitochondrial ribosomes and the synthesis of key components of the electron transport chain, without which, aerobic respiration is not possible.

To test our idea, we modified the popular yeast expression vector, p416-GPD (Müller 120), by replacing the multiple cloning site with the BioBrick prefix and suffix (pSB416-GPD). We then used overlap assembly to clone a new translational unit (BBa_K1729001), including the wild-type Kozak sequence and a yeast codon-optimized MRPS12 gene, behind the GPD promoter.

Screen captures from SnapGene®

Ultimately, we envision controlling the expression of this gene with a repressible promoter such as that of the CTR1/3 system (Labbe 149). Copper ions repress gene expression in this system. Therefore, when a higher concentration of copper is added to a culture, MRPS12 will not be expressed and aerobic respiration will not occur, forcing the cells into fermentative growth. However, a copper chelator like bathocuprione disulfonate could then be added to the system, to derepress the MRPS12 gene and allow aerobic respiration to begin again.

Such a system could be employed during industrial fermentations. Yeast growth follows an S-curve, with secondary metabolite generation occurring at the end of the exponential phase into the stationary phase. By disabling aerobic respiration, the yeast could begin generating secondary metabolites sooner and, therefore, might produce more over the course of their growth. Additionally, if fermentation products could be consumed by the yeast, such a switch could prevent this consumption to increase yields (Aerts 115).